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Research Article
 

Effect of Leishmania gerbilli Injection on Mice Immunization Against Cutaneous Leishmaniasis (CL) Caused by Leishmania major



Abedi Said, Khamesipour Ali, Izadi Shahrokh, Hejazi Hosein and Mikhak Mahin
 
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ABSTRACT

In the present study Leishmania gerbilli were used to immunize BALB/c mice against pathogenic strains of leishmania to determine whether injection of L. gerbilli in mice could protect them against later L. major inoculation. Eighty female BALB/c mice were divided by random in eight groups. Promastigotes of L. major and L. gerbilli were used. Mice were inoculated with three different doses of L. gerbilli (3x106, 2x107 and 5x107) via subcutaneous (SC) in the base of their tails or interpretoen (IP). Forty days after the first injection, all mice received the same doses as a booster. Two control groups received PBS (SC or IP) only. All BALB/c mice were inoculated subcutaneously with 2x106. Promastigotes of L. major in the base of their tails after 75 days of the first injection of L. gerbilli. When leishmania lesion developed (35 days after challenge), the size was measured and continued once a week for 12 weeks. Meanwhile, the liver and spleen samples of dead mice moved to culture media and examined for the parasite. Delaed Type Hypersensitivity (DTH) and immunoflurecent tests were used to determine results of immunization. Compared with the control group and the other groups that received different doses of L. gerbilli via IP, an evident decrease in lesion size was observed in group that received 2x107 promastigotes (p<0.05). By contrast, in those groups received L. gerbilli subcutaneously, no difference was observed through the different doses of inoculated parasite. Comparison of the inoculation styles showed that IP method caused smaller lesions than SC (p<0.05).

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  How to cite this article:

Abedi Said, Khamesipour Ali, Izadi Shahrokh, Hejazi Hosein and Mikhak Mahin, 2007. Effect of Leishmania gerbilli Injection on Mice Immunization Against Cutaneous Leishmaniasis (CL) Caused by Leishmania major. Pakistan Journal of Biological Sciences, 10: 196-198.

DOI: 10.3923/pjbs.2007.196.198

URL: https://scialert.net/abstract/?doi=pjbs.2007.196.198

INTRODUCTION

Leishmania species are known as parasitic protozoan cause of many kinds of leishmaniasis include Cutaneous (CL), Visceral and muco-cutaneous Leishmaniasis. Cutaneous Leishmaniasis (CL) is an endemic disease in many parts of the world that can cause considerable dermal disease and may result in sever disfigurement. Leishmania major and Leishmania tropica are pathogenic agents of Cutaneous Leishmaniasis (CL) in man and many other animals and transmitted by the bites of infected phlebotomine sand flies in nature.

Various efforts such as Local irradiation, cryotherapy and antileishmanial drugs, are beenig made to enhance healing of the ulcer which none of them has proved satisfactory for the treatment of CL. In the search for new ways to control of leishmaniasis there has been a major emphasis on immunization methods, for example killed amastigote vaccine against fatal L. major (Liew et al., 1987), Bacilli Calmett Gurine (BCG) as a vaccine (Stephani et al., 1993), Recombinant BCG (Connel and Medina, 1993) and different collones of L. major and L. arabica (Peters et al., 1990).

Since no effective chemotherapy on cutaneous and visceral leishmaniasis is available, many efforts focused on finding effective immunization material. In the present research Leishmania gerbilli were used to immunize animals against pathogenic strains of Leishmania to determine whether injection of L. gerbilli in mice could protect them against later L. major inoculation. Leishmania gerbilli, a kind of Leishmania species causes CL in Rhombomys opimus (but not in Meriones libycus). It first reported from Kanzo and Sinkiang in China with zoonetic cycle and shows that failes to contaminated human (Sterelkova et al., 1991). It’s Isoenzymatic pattern was studied in previous USSR and compared with other straines (Kelin and Passova, 1985). Because of such a close relationship between L. gerbilli and L. major, we suppose that early injection of L. major promastigotes may conclude further immunization in BALB/c mice.

MATERIALS AND METHODS

Animals: Eighty female Balb/C mice purchased from Pasteur Institute of Tehran and transported to laboratory of Isfahan researching center and Islamic Azad University of Falavarjan. After that, they were divided by random in eight groups following by breeding to 6-8 weeks old mice.

Parasite: Promastigotes of L. major (strain MRHO/IR/75/ER) which was isolated from Rhombomys opimus in Isfahan region (Iran) and L. gerbilli (strain MRHO/CN/60/GERBILLI/LON25) that was obtained from Tarbiat Modarres University (Tehran-Iran), were used. Promastigotes cultured in RPMI 1640 medium (mixed by 10% fetal calf serum, 100 μg mL–1 Streptomycine, 50 μg mL–1 Gentamycine, 100 IU mL–1 Penicillin) and obtained in stationary phase of culture as well as washed three times in PBS.

Immunization: Mice were inoculated with three different doses of L. gerbilli (3x106, 2x107 and 5x107) via Sub Cutaneous (SC) in the base of their tails or Inter Pretoen (IP). Forty days after the first injection, all mice received the same doses as a booster and two control groups received PBS (SC or IP) only.

Challenge: In order to study the role of immunization in Cutaneous leishmaniasis, all Balb/C mice were inoculated subcutaneously with 2x106 Promastigotes of L. major in the base of their tails after 75 days of the first injection of L. gerbilli. When leishmania Lesion developed (35 days after challenge), the size was measured in two dimension at right angles to each other with a caliper gauge and continued once a week for 12 weeks. Meanwhile, the liver and spleen samples of dead mice moved to culture media and examined for the parasite.

Statistical analysis: The Tukey-HSD test was used with SPSS software (Microsoft excel version 9) to analyze the data.

RESULTS

In this study, animals (L. gerbili-infected BALB/c mice) were injected by L. major at 75th day and five weeks later the nodule transformed into an ulcer which increased in size.

Fig. 1: Ulcer size in groups 1, 2, 3 and control

Fig. 2: Ulcer size in groups 4, 5, 6 and control

Compared with the control group and the other groups that received different doses of L. gerbilli via IP, an evident decrease in lesion size was observed in group that received 2x107 promastigotes (p<0.05), (Fig. 1). By contrast, in those groups received L. gerbilli subcutaneously, no difference was observed through the different doses of inoculated parasite (Fig. 2). Comparement of the inoculation styles, showed that IP method caused smaller lesions than SC (p<0.05). In other experiment, mice which received 3x106 or 5x107 L. gerbilli promastigotes subcutaneously or by interpretoen, no significantly difference was seen in lesion size.

DISCUSSION

In this study we used L. gerbilli promastigote injection as a vaccine to protect BALB/c mice against later L. major infection. Isoenzyme pattern of L. gerbilli is similar to L. major P-K, PEP-1 and L. tropica ES (Xu et al., 1984), so we suspected that L. gerbilli might presents as a immunized agent to protect BALB/c mice. Pramastigotes of different strains of Leishmania was used before. Barrel used L. mexicana amazonensis Promastigotes; he used three different doses of parasite (5x107, 2x107 and 3x107) and showed when doses of antigen increased, it lead to more protection (Burrel et al., 1987). In other study they used five different doses (2x104, 2x105, 2x106, 2x107 and 2x108) of radiated L. major to protect mice against L. major and reported that although protection in all groups were seen; but the better results were seen in 2x107 promastigote derived (Liew et al., 1985). This results are similar to that we showed here. Liew at all reported that subcutaneously inoculation of 2x104, 2x105, 2x107 and 2x108 radiated amastigotes of L. major in BALB/c mice induced bigger lesions compared with control groups and doses of parasite had not been effect the size of lesions (Liew et al., 1985). In this study no difference were seen in lesion size of mice derived different doses of L. gerbilli subcuteneousely. Although the mortality rate in mice was increased as the doses of parasite derived was increased.

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